Impacts of Diversification of Cytochrome P450 on Plant Metabolism

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Biol. Pharm. Bull. 35(6) 824–832 (2012)

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Current Topics

The 50th Anniversary and New Horizons of Cytochrome P450 Research: Expanding Knowledge on the Multiplicity and Versatility of P450 and Its Industrial Applications Impacts of Diversification of Cytochrome P450 on Plant Metabolism Masaharu Mizutani Functional Phytochemistry, Graduate School of Agricultural Science, Kobe University; 1–1 Rokkodai, Nada, Kobe 657–8501, Japan. Received January 25, 2012 Cytochrome P450 monooxygenases (P450s) catalyze a wide variety of monooxygenation reactions in primary and secondary metabolism in plants. The share of P450 genes in each plant genome is estimated to be up to 1%. This implies that the diversification of P450 has made a significant contribution to the ability to acquire the emergence of new metabolic pathways during land plant evolution. The P450 families conserved universally in land plants contribute to their chemical defense mechanisms. Several P450s are involved in the biosynthesis and catabolism of plant hormones. Species-specific P450 families are essential for the biosynthetic pathways of phytochemicals such as terpenoids and alkaloids. Genome wide analysis of the gene clusters including P450 genes will provide a clue to defining the metabolic roles of orphan P450s. Metabolic engineering with plant P450s is an important technology for large-scale production of valuable phytochemicals such as medicines. Key words

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P450; metabolic engineering; terpenoid; phytohormone

P450S IN THE PLANT KINGDOM

More than 5000 sequences have been stored in a plant P450 database.1) The number of P450 genes in Arabidopsis (Arabidopsis thaliana) is 245, and P450 is the third biggest family gene in Arabidopsis.1) The numbers of P450 genes in several other plants are 316 in grapevine (Vitis vinifera), 332 in soybean (Glycine max), 312 in poplar (Populus trichocarpa), 334 in rice (Oryza sativa), and 372 in sorghum (Sorghum bicolor), which are estimated to represent up to 1% of the total gene annotations of each plant species.2,3) Transcriptome analyses are also contributing to the vast increase in identified P450 sequences from various plant species (http://compbio.dfci. harvard.edu/tgi/plant.html). However, the functions of most of these are still unknown. For instance, in Arabidopsis, the number of functionally characterized P450 genes is about 60, which means that more than 70% of its 245 P450 genes still remain to be characterized.4) Plant P450s have been shown to participate in a variety of biochemical pathways to produce primary and secondary metabolites such as phenylpropanoids, alkaloids, terpenoids, lipids, cyanogenic glycosides, and glucosinolates, as well as plant hormones.4) The diversification of P450 has had a significant biochemical impact on the emergence of new metabolic pathways during the evolutionary process of land plants.

2. DIVERSIFICATION OF P450 GENES IN LAND PLANTS The driving force of plant P450 diversification is closely linked to the survival strategy of land plants, conferring advantages in the continuous evolutionary competition and mutualism with other organisms. The role of plant P450 can

be discussed in relation to the following categorized reactions and metabolisms: category I, essential reactions conserved in the plant kingdom; category II, core metabolisms conserved in all land plants; category III, essential metabolisms which emerged during the evolution of flowering plants (e.g., plant hormone homeostasis); category IV, secondary metabolisms unique to individual species.

3. P450S PARTICIPATING TO CORE METABOLISMS (CATEGORIES I AND II) The P450s of category I include 3 families (CYP51, CYP710, and CYP97), which are conserved in the plant kingdom from green alga to vascular plants. CYP51G and CYP710A encode obtusifoliol 14α-demethylase and sterol 22-desaturase, respectively, which are essential for sterol biosynthesis.5,6) The CYP97 family functions in xanthophyll biosynthesis, and Arabidopsis CYP97A3 and CYP97C1 catalyze the hydroxylation of β- and ε-rings, respectively, of carotenoids in the biosynthetic pathway of xanthophylls, which are key components in photosynthetic complexes for light-harvesting and photoprotection.7,8) These reactions are likely essential for the normal growth and development of plant cells. The P450s of category II consist of several P450 families conserved in land plants. During the adaptation to terrestrial conditions, plants have been required to develop the synthesis of biopolymers covering their surfaces to protect against dryness and UV radiation. In addition, land plants have needed to produce various defense chemicals against herbivores and pathogens. These chemical defense mechanisms are key strategies that allow land plants to survive and expand their habitats on earth. The hydroxylation of cinnamates is the key reaction in the phenylpropanoid pathway, and is essential for

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the biosynthesis of a variety of phenolic compounds, having such various functions as structural components (e.g. lignin and suberin), UV protectants (flavonoids), antioxidants (polyphenols), antimicrobials (coumarins, lignans, isoflavonoids), and flavors (benzenoids, phenylpropenes). The CYP73A and CYP98A subfamilies are conserved in all land plants examined to date; the CYP73A subfamily contains only cinnamate 4-hydroxylases,9,10) and CYP98A catalyzes the 3′-hydroxylation (meta-hydroxylation) of coumaroyl esters.11,12) The conservation of the para- and meta-hydroxylation of cinnamates during land plant evolution suggests strong selective pressures for the synthesis of phenylpropanoids. Hydroxylation of fatty acids is also a key biochemical reaction involved in constructing complex biopolymers such as cutin and suberin. Cutin is one of the essential components of the epidermal cuticle, consisting of ω-hydroxylated fatty acids (mainly C16 and C18 fatty acids) and glycerol.13,14) Suberin, contributing to the diffusion control of water and solute across internal root tissues and in periderms, is composed of a polyaliphatic (fatty acid-derived) domain cross-linked by ester bonds to a polyaromatic domain.13) These fatty acid-derived biopolymers play crucial roles in controlling water evapotranspiration, protecting the plants against UV and pathogens, and regulating cell fusion during organ growth.15,16) Fatty acid hydroxylases are encoded by several different P450 families and catalyze the hydroxylation of fatty acids at the ω, ω-1, or ω-2 position.17,18) The CYP86 family is known to function in the ω-hydroxylation of fatty acids with chain lengths ranging from C10 to C18.15,16,19) In addition, the CYP94 family members are known to catalyze the ω-hydroxylation of long chain fatty acids neccesary for cutin and suberin biosynthesis.17) The CYP86 and CYP94 families are conserved throughout land plants and are involved in producing the essential biomolecules covering plant surfaces, including aerial surface, roots, and pollen. The CYP703 family is also conserved in all the land plants. Arabidopsis CYP703A2 catalyzes the in-chain hydroxylation of mid-chain fatty acids, which is the essential building block of exine.20) This material is in the outer walls of pollens and consists of a mixture of biopolymers containing hydroxylated fatty acids and phenylpropanoids.20) In Physcomitrella and Selaginalla, spores can be regarded as an equivallent to pollens, and the CYP703 family in lower plants is likely involved in the synthesis of sporopollenin, a major component of spore walls.

4. P450S IN PHYTOHORMONE HOMEOSTASIS (CATEGORY III) Several P450s are known to contribute to the biosynthesis and catabolism of phytohormones (Fig. 1). CYP735As contribute to the hydroxylation of isopentenyladenine riboside 5′-monophosphate to form trans-zeatin riboside 5′-monophosphate in cytokinin biosynthesis.21) Abscisic acid (ABA) is a phytohormone controlling seed dormancy, stomatal closure, and environmental stress tolerance. A key step of the ABA catabolism is the 8′-hydroxylation by CYP707A 2,22,23) (Fig. 1A). Gibberellins (GAs) are diterpenoid phytohormones regulating seed germination and shoot growth, and several P450s contribute to GA biosynthesis. CYP701A catalyzes the 3-step oxidations of ent-kaurene to form ent-kaurenoic acid (Fig. 1B).24) CYP88A catalyzes the 3-step oxidations of ent-karenoic

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acid to form GA12.25) The 13-hydroxylation converting GA12 to GA53 is also a P450-dependent reaction. Arabidopsis CYP714A1 has been reported to catalyze the 13-hydroxylation of ent-kaurenoic acid to form steviol,26) and rice CYP714D1 is a 16α,17-epoxidase involved in GA inactivation.27) Arabidopsis CYP714A1 and CYP714A2 are redundant genes for GA inactivation.28) These facts suggest that C-13 hydroxylation is an inactivation process of GA rather than a biosynthetic reaction. Brassinosteroids (BRs) are plant steroid hormones functioning in the regulation of growth and development, and castasterone and brassinolide, which are the most active BRs, are biosynthesized from campesterol.29,30) The genetic and chemical analyses of BR-deficient dwarf mutants led to the identification of several P450 genes necessary for BR biosynthesis in various plants. The functional analysis of these P450s has revealed that the biosynthetic pathway of brassinolide consists of successive oxidations of the side chain and A/B ring of campesterol by CYP85A, CYP90A, CYP90B, CYP90C, CYP90D, and CYP724B31–37) (Fig. 1C). Phylogenetic analysis indicated that these P450s are classified into one clade, the CYP85 clan.30) This implies that the BR-biosynthetic P450 genes have evolved from a common ancestral gene, and that their duplication and divergence result in the emergence of a series of P450-dependent reactions necessary for the sequential modification of the steroid structure. The CYP734A subfamily is known to be involved in BR catabolism. Arabidopsis and tomato CYP734As encode a 26-hydroxylase of brassinolide and castasterone,38–40) while rice CYP734A shows broad specificity to the intermediates of BR biosynthesis and catalyzes 3-step successive oxidations at C2641) (Fig. 1C). Oxylipins are bioactive lipid metabolites formed by the oxygenation of polyunsaturated fatty acids. Jasmonic acid (JA) is a representative plant oxylipin, which controls pollen-tube development and stress response. JA is formed from 13-hydroxyperoxyoctadecatrienoic acid (13-HPOT) by allene oxide synthase (AOS), which is a P450 that belongs to the CYP74A subfamily.42) JA-isoleucine has been identified as an active ligand for the JA signaling pathway, and JA-isoleucine is inactivated by CYP94B3 and CYP94C1 (Fig. 1D).43–45) Green leaf volatiles are also produced from 13-HPOT by hydroperoxide lyase (HPL), a P450 belonging to the CYP74B subfamily.46) CYP74A (AOS) and CYP74B (HPL) are atypical P450s requiring neither oxygen nor an electron for the catalytic activity. Recently the three-dimensional structures of CYP74A (AOS) from Arabidopsis and Parthenium argentatum have been determined, and the reaction mechanisms of AOS and HPL have been clarified..47,48) Strigolactones (SLs) are a group of carotenoid-derived signal compounds which are secreted from roots and induce the seed germination of root parasitic plants such as Striga and Orobanche species.49) SLs are shown to act as root derived branching factors for symbiotic interaction with arbuscular mycorrhizal fungi.50) SL-deficient mutants show an increase in shoot branching, and SLs have been found to act as a new class of plant hormone, which inhibits shoot branching and controls axillary bud outgrowth.51–53) Characterization of these mutants demonstrates that SL biosynthesis is dependent on the genes encoding carotenoid cleavage dioxygenases CCD7 and CCD8.54) Arabidopsis CYP711A1 (MAX1) is involved in SL biosynthesis and likely acts downstream of the CCDs, but the catalytic function of CYP711A1 remains to be characterized55)

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Fig. 1.

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P450s Involved in Biosynthesis and Catabolism of Phytohormones

(A) CYP707A in abscisic acid catabolism; (B) P450s in gibberellin biosynthesis and catabolism; (C) P450s in brassinosteroid biosynthesis and catabolism; (D) P450s in jasmonyl-isoleucine catabolism; (E) CYP711A in strigolactone biosynthesis.

(Fig. 1E).

5. P450S IN SECONDARY METABOLISM (CATEGORY IV) Plants are characterized by their ability to produce a wide variety of organic compounds, or so-called secondary metabolites, and they comprise more than 200000 natural plant products. The huge complexity and diversity of secondary metabolites is due to the diversity of the genes encoding the enzymes responsible for these metabolisms. Of these genes, the diversified P450 genes are the most important. Many plant species synthesize specific phytochemicals such as taxol, benzylisoquinoline alkaloids, isoflavonoids in Faboideae, and glucosinolates in Brassicaceae. The production of such metabolites often depends on many species-specific P450s, and the evolutional diversification of these P450s might be essential for establishing new metabolic pathways branched from the pre-existing metabolic pathways. This section describes recent discoveries of P450 genes in the biosynthesis of taxol, benzylisoquinoline alkaloids, and triterpenoid saponins as representative examples. Taxol is one of the taxane diterpenoids (taxoids) produced by Taxus species and is an effective anticancer agent. The biosynthesis of taxol requires at least 19 steps, beginning

with the cyclization of geranylgeranyl diphosphate to taxa4(5),11(12)-diene by taxadiene synthase. This precursor, having a taxane-core structure, is modified by sequential metabolisms including eight P450-mediated reactions. Seven P450s have been identified as 2α-, 5α-, 7β-, 9α-, 10β-, 13α-, and 14β-hydroxylases of taxoids56,57) (Fig. 2A). These P450s showed over 70% sequence identity among them, but the similarity to other plant P450s are less than 35%. These P450s are classified as the CYP725 family, occurring only in Taxus species. Since taxoids have antimicrobial activities, their production might have expanded the survival advantage of Taxus species, and this evolutionary pressure will be a driving force for differentiating CYP725 to generate a series of P450s participating in the modification of the taxadiene-core structure with high regio-specificity. Benzylisoquinoline alkaloids (BIAs) are a large group of alkaloids including many pharmacologically useful compounds, e.g., morphine, codeine, berberine, and sanguinarine. BIAs are biosynthesized from tyrosine via the key precursors (S)-norcoclaurine and (S)-reticuline through the modification by P450s and methyltransferases. Several P450s in the CYP80 and CYP719 families contribute to the BIA biosynthesis (Fig. 2B). CYP80A1 of Berberis stolonifera catalyzes the intermolecular C-O phenol-coupling to form berbamunine,58) and CYP80B is the 3′-hydroxylase of N-methylcoclaurine.59) CYP80G2

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Fig. 2.

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P450s Involved in the Biosynthetic Pathways of Secondary Metabolites

(A) P450s in taxol biosynthesis; (B) P450s in benzylisoquinoline biosynthesis; (C) P450s in glycyrrhizin biosynthesis.

of Coptis japonica catalyzes the intramolecular C-C phenol coupling reaction converting (S)-reticuline to corytuberine.60) The CYP719A cDNAs were isolated from C. japonica and Eschscholzia californica, producing BIAs with methylenedioxy bridge, and CYP719As actually catalyze the formation of this structure.61) CYP719B1 of Papaver somniferum is the salutaridine synthase catalyzing C–C phenol coupling in morphine biosynthesis.62) Thus, CYP80s and CYP719s are the P450 families characteristic of alkaloids-producing plants, and they are not found in the genomes of Arabidopsis, rice, poplar, and lower plants. These results suggest that the evolution of these P450 families is closely related to the diversification of BIAs. Saponins are a group of glycosides of triterpenoids and steroids conjugated with oligosaccharide at C-3. Saponins are considered to act as the defense mechanism against pathogens and herbivores, and some of them have been used as medicines and their materials. The diversity of saponins is dependent on the mode of cyclization of 2,3-oxidosqualene

and subsequent modification of the triterpenoid or sterol rings by oxygenation and glycosylation. Although the roles of 2,3-oxidosqualene cyclases and glycosyltransferases in saponin biosysthesis have been established,63,64) P450s responsible for the modification of triterpenoids and steroids in saponin biosynthesis still remain to be characterized. Soybean accumulates soyasaponins, and its CYP93E1 was identified as β-amyrin C-24 hydroxylase, involved in soyasaponin biosynthesis.65) Glycyrrhizin accumulated in the root and stolon of Glycyrrhiza species exhibits a wide range of pharmacological activities such as anti-inflammatory, antiallergic, and antiviral activities. The biosynthetic pathway of glycyrrhizin involves the oxidation of β-amyrin at C-11 and C-30 by P450s to form glycyrrhetinic acid (Fig. 2C). CYP88D6 of G. uralensis was identified as β-amyrin 11-oxidase, and the yeast cells coexpressing CYP88D6 and β-amyrin synthase could produce 11-oxo-β-amyrin in vivo.66) In addition, homology-based PCR cloning with soybean CYP93E1 revealed that CYP93E3 from

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Fig. 3.

Genomic Organization of CYP716As in the Grapevine Chromosomes

(A) gene clusters of CYP716As and β-amyrin sythase-like (bAS-like) genes on chromosome 4 and 11. (B) Reaction scheme of C28-oxidation of β-amyrin by CYP716A.

G. uralensis is also identified as β-amyrin 24-hydroxylase. Recently, the second P450, CYP72A154, has been identified as the C-30 oxidase involved in forming glycyrrhetinic acid.67) Thus, G. uralensis contains three different P450 families (CYP93E, CYP88D, and CYP72A) to oxidize β-amyrin at distinct positions. This fact indicates that these P450 genes originate from phylogenetically distant ancestral P450s to produce β-amyrin-derived saponins in G. uralensis.

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P450 GENE CLUSTERS IN THE GENOME

Recent genomics studies have revealed the occurrence of operon-like gene clusters in eukaryotic genomes, and such gene clusters are sometimes expressed in an integrated manner to achieve certain biological functions.68–70) In plants, gene clusters including P450s are known to be involved in speciesspecific metabolisms. Typical examples of such P450 involvement in gene clusters are reported for thalianol biosynthesis by Arabidopsis (CYP705A and CYP708A),71) benzoxazinoid biosynthesis by maize (CYP71C),72) avenacin biosynthesis by oat (CYP51H),73) and momilactone biosynthesis by rice (CYP71Z, CYP76M, and CYP99A).74,75) Neighboring genes tend to be co-regulated to constitute a set of harmonized metabolic functions.76,77) Evolutional processes of gene clustering have not been clarified, and co-regulated genes are not explained simply by the known metabolic pathways. Finding co-regulated clustered genes might provide a clue to allow us to propose new hypotheses. For example, such clustering genes often contain genes encoding the enzymes participating in known metabolisms, and a target analysis to identify the function of a novel P450 gene included in the clustered genes might be designed. A typical example of such targeted analysis to determine the function of P450s is shown in Fig. 3.78) CYP716A17, which is located in tandem with CYP716A15 on chromosome 11 of grapevine,

is clustered to a β-amyrin synthase-like (bAS-like) gene. CYP716A19 and CYP716A20 are also located adjacent to bASlike genes on chromosomes 4 and 11, respectively. Functional characterization of CYP716A15 and CYP716A17 showed that they catalyze the successive oxidation at C-28 of β-amyrin to form oleanolic acid, indicating the functional correlation of CYP716As with bAS-like genes in the gene clusters.

7. METABOLIC ENGINEERING FOR PRODUCTION OF USEFUL PHYTOCHEMICALS Many phytochemicals are used as medicines and their materials. Most of them are produced by specific plant species, and the amount available from natural sources is limited. To ensure their stable supply, production by metabolically-engineered microorganisms is expected. Since P450s play crucial roles in the biosynthesis of phytochemicals, identification of these P450s and development of their heterologous expression are important for metabolic engineering to produce useful phytochemicals. In the case of metabolic engineering with microbial systems, the biosynthetic pathway of phytochemicals is usually divided into two phases: Phase I for the production of key intermediates through primary pathways, and Phase II for their modification by secondary enzymes including P450s. For example, the metabolic engineering of taxol production by Escherichia coli is depicted in Fig. 4A. Effective production of taxadien-5α-ol (58 mg/L) by E. coli has been done through the optimization of phase I to produce taxadiene in high yield (1 g/L), followed by its hydroxylation with a fused enzyme of CYP725A4 and P450 reductase as phase II.79) This is a successful example of the production of the taxol precursor, although at least six additional P450s and acyltransferases are necessary for converting taxadien-5α-ol to baccatin III, the material required for the semi-synthetic production of taxol.

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Fig. 4.

Strategies for Producing Taxol and Artemisinin by Metabolic Engineering of Microbial Systems

In the biosynthetic pathway of artemisinin, an anti-malaria agent, amorpha-4,11-diene produced by amorphadiene synthase (ADS) in phase I metabolism undergoes phase II metabolism by CYP71AV1 to form artemisinic acid, the material required for the semi-synthetic production of artemisinin (Fig. 4B). The microbial systems expressing the cDNAs of ADS, CYP71AV1, and P450 reductase produced artemisinic acid in both yeast (115 mg/L)80) and in E. coli (105 mg/L).81) Artemisinic acid and artemisinin were produced in tobacco by plant metabolic engineering.82,83) The leaves of Nicotiana benthamiana infected by agrobacteria transformed by the cDNAs encoding ADS, hydroxymethylglutaryl (HMG)-CoA reductase and farnesyl pyrophosphate synthase accumulated amorpha-4,11-diene, and co-infiltration of the agrobacteria containing CYP71AV1 cDNA caused an accumulation of artemisinic acid-12-β-diglucoside (39.5 mg/kg fr. wt). Bioactive artemisinin was also produced in N. tabacum83) by transfection with a mega-vector carrying the cDNAs of CYP71AV1, P450 reductase, ADS, artemisinic aldehyde reductase and HMGCoA reductase. This transgenic tobacco accumulated artemisinin (6 µg/g dry weight), indicating that dihydroartemisinic aldehyde produced by the mega-vector is non-enzymatically converted to the final metabolite artemisinin. Thus, the whole pathway of artemisinin biosynthesis can be reconstructed in the transgenic plants by metabolic engineering. A metabolically engineered system assembling several microbial and plant enzymes in E. coli was constructed for producing reticuline from dopamine, and succeeded in producing (S)-reticuline with a yield of 55 mg/L.84) Co-cultivation of this reticuline-producing E. coli with yeast cells expressing C. japonica CYP80G2 (corytuberine synthase) could produce scoulerine, a protoberberine alkaloid, with a yield of 8.3 mg/L.84) This result suggests that metabolically engineered microbial cells assembling microbial and plant enzymes may be useful for producing valuable phytochemicals and newly designed chemicals.

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CONCLUDING REMARK

P450s constitute a huge gene group, accounting for more than 1% of each plant genome. The diversification of plant P450 is one of the dominant driving forces of the diversity of

phytochemicals. The number of P450 genes identified in plant genomes will be increased rapidly, and the diversity and complexity of phytochemicals will be further disclosed using plant metabolomics. The cooperation of transcriptomics and metabolimics will provide clues to finding novel functions of P450s. Genomics will provide new information for understanding the reason for the diversity of plant P450s, and analysis of gene clusters including P450 genes will provide a clue to unraveling novel metabolic activities of plant P450s. Plant P450s catalyze various reactions underlying key structures for biological activities of phytochemicals; therefore, characterization of these P450s is essential for the biotechnological production of valuable phytochemicals. Metabolic engineering systems that express the biosynthetic genes as well as the regulatory genes will provide great advantages for large-scale production of phytochemicals in the near future, and integration of multiple P450-dependent reactions into the systems poses a big challenge for constructing the metabolic pathways producing valuable phytochemicals. Finally, the expansion of structural information of plant P450s is necessary for elucidating their reaction mechanisms, and such developments will enable us to use abundant and various plant P450s as desired bioconversion systems.

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